The HIV-1 protease is becoming the focus of intense study both as a key enzyme in the life cycle of HIV-1 and as a critical target for the development of an effective antiviral. The protease plays a central role during virion assembly as it cleaves the viral Gag and Gag/Pol precursors. Ordered and near complete cleavage of these precursor proteins is required for the efficient budding of virus particles and the formation of infectious virions. This central role in the virus life cycle makes the protease a biologically important protein, and its small size and well characterized catalytic activity make it amenable to a variety of biochemical analyses. In addition, it is possible to do structural analysis of the protease, making all of the major tools for protein characterization available for the study of the viral protease. We currently have a library of over 500 characterized protease mutants, and this library is the starting point for selecting mutants for further detailed study. In addition, experimental approaches are available to study the protease as a model for protein evolution, and to study the role of and control of ordered processing of the viral precursor proteins during particle assembly. Taken together, these studies provide fundamental insights into the molecular details of protein function for this critical viral protein. Specifically, I propose the following: i) Extend our ongoing structure-function analysis of the HIV-1 protease, focusing on issues of substrate specificity, protein stability, and structure determination; ii) Examine how protease sequences are evolving in the different clades of HIV-1 and the role of compensatory mutations in fixing sequence changes; iii) Explore the role of ordered processing of the Gag and Gag/Pol precursors during virion assembly and the determinants that regulate ordered processing.